Gluconacetobacter liquefaciens and Gluconacetobacter xylinus share very similar phenotypic characteristics. They are differentiated by the production of a reddish-brown water-soluble pigment of the former and cellulose production of the latter. However, the loss of the two distinguishing features questions the separate standings of the two species. The DNA base composition and the DNA relatedness of strains of the two species, including other established species of acetic acid bacteria, were determined. G. liquefaciens strains had the higher guanine-plus-cytosine content (G+C content) in DNA, ranging from 63.5 to 66.9 mol%, and G. xylinus had the lower range, from 59.4 to 63.2 mol%. DNA hybridization revealed a low level of DNA similarity between the two species. G. liquefaciens strains produced 2,5-diketogluconic acid and pyrones from glucose, and G. xylinus strains produced 5-ketogluconic acid. From these results, it is unequivocal that G. liquefaciens is a distinct species from G. xylinus.